Process for the isolation of cholesterol from wool-wax alcohols



Patented Sept. 7, 1954 PROCESS FOR THE ISOLATION OF CHOLES- TEROL FROM WOOL-WAX ALCOHOLS Colin Leslie Hewett, London, England, assignor to Organon Laboratories Limited, London, England, a British company No Drawing. Application June 2'7, 1951, Serial No. 233,955

Claims priority, application Great Britain July 21, 1950 6 Claims.

The present invention relates to a new or improved process for the isolation of cholesterol (which is an important material for the manufacture of sex harmones) from the non-saponifiable component of wool grease known as woolwax alcohols.

In a copending application of mine, Serial No. 78,896, now Patent No. 2,650,929,. a process of this character is described wherein after separating unwanted iso-cholesterol by known means, separation of the wanted cholesterol is performed either by selective crystallisation of the cholesterol or by selective dissolution of wax-like impurities, in either case in the presence of acetic acid or other simple fatty acid.

This process gave a better yield of cholesterol, in a high state of purity, than was previously possible, but there was some loss of the wanted cholesterol in the extraction of the iso-cholesterol (which consists mainly of agnosterol and lanosterol) since this was performed by the usual process of re-crystallising the starting materials from large volumes of methanol at elevated temperature; and moreover this process necessitated working with relatively large volumes of liquids which often led to difliculties in the treatment plant used. The main object of the present invention is to provide a process for the separation of cholesterol from wool-wax alcohols whereby a still higher yield of cholesterol in a high state of purity may be obtained. A further advantage is the isolation of iso-cholesterol in substantial amounts and of high purity.

In accordance with the present invention a process for separation of cholesterol from woolwax alcohols, comprises separating iso-cholesterol substantially free from cholesterol by crystallising the iso-cholesterol from a mixture of benzene or its lower homologues and methanol, and thereafter purifying the cholesterol from wax-like impurities by selective crystallisation from a simple fatty acid such as acetic acid or selective dissolution of the impurity by a simple fatty acid, in either case under carefully regulated temperature conditions.

In carrying out the present invention the woolwax alcohols are melted at about 60 C. and dissolved in 2 volumes of a mixture of benzene (or its lower homologues such as toluene or xylene) and methanol and allowed to cool when the iso-cholesterol separates in fine needles.

The proportion of benzene in the benzenemethanol mixture may lie between and 60% by volume, the temperature for crystallisation being appropriately adjusted according to the benzene content. The best yield of cholesterol (Cl. 260397.Z5)

2 (and of iso-cholesterol) is obtained by a mixture containing about 20% benzene, the iso-cholesterol being allowed to crystallize and being filtered off at about 30 C. If the benzene is increased to about 50 to the temperature of crystallisation and filtering should be lowered to about 20 to 25 C., with consequent convenience in practical operation, although in this case the amount of iso-cholesterol is somewhat reduced. It should be noted that the use of methanol in this way is of great advantage as the iso-cholesterol is precipitated in crystalline form, whereas other alcohols tend to produce amorphous material. The iso-cholesterol forming 5 to 10% of the original wool-wax alcohols is then filtered off and the filtrate distilled to dryness. When this residue is crystallised from a lower fatty acid such as acetic or propionic acid at 30 to 35 C. by a method analogous to that described in my copending application Ser. No. 78,896, filed February 28, 1949, now Patent No. 2,650,929, cholesterol selectively separates readily, and on being recrystallised, if desired, from alcohol (e. g. industrial methylated spirit), a pure product, in yields of 10 to 20% is obtained. Alternatively the acetic or propionic acid solution may be allowed to cool to room temperature and the wax-like material selectively dissolved by gently warming to 30-35 C. with stirring; the yield being of the same order as above. The different temperatures mentioned above compared with the aforesaid specification result from the different proportion of iso-cholesterol in the mass under treatment. Experience has shown that the vital range is, in most cases, the range of 30 to 32 C., over which the wax-like'impurity presumably can exist in the form of a supersaturated solution. Hence on gradual cooling of the mass the cholesterol crystallises, this action being preferably facilitated by seeding, crystallisation being substantially complete by 30 to 31 0., below which temperature the impurity is deposited. This latter temperature will vary slightly from batch to batch. In point of fact crystallisation starts at about 50 C. so that a higher final temperature may be used if a smaller yield of the wanted cholesterol can be tolerated. Likewise in the selective dissolution procedure, on raising the temperature to about 30 to 31 C. the wax-like residue is dissolved while the wanted cholesterol is substantially unaffected; the temperature could in fact be raised to as much as 45 C. if a smaller yield can be tolerated. It may be stated, however, that a commercially advantageous yield is obtained if the temperature lies within the range of 30 to 35 0., this applying both for selective crystallisation of the wanted cholesterol and for selective dissolution of the impurity. This yield is higher than that possible by any other known processes and the cholesterol is of a high state of purity. This process also has the advantage that the reacting mixtures occupy a volume of slightly more than one-third that of the earlier processes, thus enabling larger batches to be carried out.

The following examples will. serve to explain the invention more fully:

Example 1 200 kg. of wool-wax alcohols is mixed with 500 l. of a mixture of 60% benzene and 40% methanol (v./v.), brought to the boil (57 Cl) and refluxed until all the wax is dissolved. The mixture is cooled to 50 C. with agitation and seeds of iso-cholesterol added and allowed to stand and to cool over the next two days to a temperature of 30. to 31 C. By seeding with iso-cholesterol and cooling over two days, larger crystals are formed, greatly simplifying filtration. On the third day the mixture is filtered under vacuo and the crystals collected. If filtration is effected at a temperature above 30 C., the filtration is rapid as the wax-like impurities have not separated from the solution. If, however, the temperature exceeds 35C., cholesterol is lost. The filtrate is distilled at atmospheric pressure until the temperature reaches about 110 C.; the amount of solvent collected in the receiver should be about 450 to 500 1.; this is returned for retreatment of the starting material. Two batches of solid material are collected from the still and final traces of the solvent are removed in vacuo. Yield of crude product 360 kg. from the two batches.

The purification of the above crude product is then effected by one of the methods described in the aforementioned copending application of mine, Ser. No. 78,896, now Patent No. 2,650,929, and the following particulars are given as a preferred method for such further purification based on the selective crystallisation method under carefully regulated temperature conditions.

In this further treatment 3601. of glacial acetic acid are mixed with the 360 kg. of crude product and the temperature is adjusted to about 60 C. and the mixture stirred to form a homogeneous solution. The temperature is reduced to 50 C. and seeds of cholesterol are added. The mixture is allowed to cool, with occasional stirring, to 32 C. over' two days. With the temperature held at 32 C'. the crystal masses are broken up and the solid material filtered off at a temperature held at about 32' to 35 C. to prevent separation of Wax. The purified cholesterol on the filter is washed with 150 l. of acetic acid at 32 C; and is dried in vacuo. Yield at this stage is 60 to 80kg.

Further purification, if desired, may be effected by re-crystallisationfrom industrial methylated spirit. The aforesaid 60 to 80 kg. of the solid product is refluxed with 240 l. of industrial methylated spirit for half an hour and then allowed to cool and crystallise over-night. The crystallised cholesterol is filtered and washed with industrial spirit. Final yield: 60' to 70 kg. Melting point: 148 to 149 C. Specific rotation in chloroform: 39.

litres of methanol-benzene mixture containing 40% methanol, at the boil. After cooling down overnight to 20 C. the iso-cholesterol ('72 gms.) was filtered off and washed with 100' cc. of the methanol-benzene mixture. The filtrate was distilled down to dryness and the residue dissolved in 1 l. of propionic acid. The solution was then allowed to cool slowly, with occasional agitation, to 30 C. during 4 to 5 hours. The cholesterol which crystallised was filtered off and washed with a little propionic acid, then with water and dried.

Yield'.185gms. M. P. 146-14'7 Example 3 1 kg. of wool-wax alcohols was treated as in Example 2: to remove isocholesterol, dissolved in 1 1'. of propionic acid and allowed to cool down to room temperature overnight. The solid mass was then slowly warmed with stirring to 32 C. when the wax dissolved leaving the crystalline cholesterol undissolved. The cholesterol was filtered off, washed with warm propionic acid and then water and dried.

YieZd.180 gms. M. P. 1465-1475 C.

Example 4 500 gms. of wool-wax alcohols were dissolved in l l. of a mixture of methanol-benzene mixture containing methanol. The solution was slowly cooled to 30 C., kept at this temperature for 2 hours and then the crystalline iso-cholesterol filtered off and Washed with cc. of the same methanol-benzene mixture at 30 C. The yield of iso-cholesterol was 57 gms. The filtrate on cooling to 15 C. deposits a waxy material which may be filtered off if the wax is desired but filtration is difficult. The filtrate containing the wax was distilled to dryness and the residue dissolved in 500. cc; of glacial acetic acid and slowly cooled to: 30 C.; after being maintained at this. temperature for 2 hours the crystals of cholesterol were filtered oil, washed with a little glacial acetic acid at 30 C., then washed with water and dried.

Example 5 1 kg. of wool-Wax alcohols was dissolved in 2%,; l. of a boiling benzene-methanol mixturecontaining 40% methanol. The solution was cooled slowly to 20 C. and kept. at 20 C. for a further 12 hours. The iso-cholesterol (60 gms.) was then filtered off and the filtrate distilled to dryness. and the residue dissolved in- 1 litre of glacial acetic acid, seeded with cholesterol and allowed to cool slowly toroom temperature with occasional agitation. After 24 hours themass was gently warmed to-- 3'2 C. with. agitation, until all the wax-like material had dissolved and the undissolved crystals of cholesterol were filtered off, washed with a little glacial acetic acid at 32 and then water. After drying the yield was 191 gms. M. P. 147-1485 C. Recrystallisation of industrial methylated spirits gave 183 gms. M. P. 149-149.5 C.

Example 6 500 gms. of wool-wax alcohols weredissolved in a boiling mixture of 550 cc. of toluene and 45000. of methanol. After allowing, to. cool to 20 C. overnight, the iso-cholesterol (39 gms.) was filtered oil and; dried. The. filtrate. was distilled to dryness at reduced pressure and. the residue dissolved in 500 cc. of glacial. acetic acid. The solution was allowed to cool slowly to. 30 kept at this temperature. for 2-3 hours and the crystalline cholesterol filtered off, washed with warm acetic acid, then water and dried.

Yield-96 gms. M. P. 147-148 C.

Example 7 500 gms. of wool-wax alcohols were dissolved in a boiling mixture of 500 cc. of xylene and 500 cc. of methanol. After allowing to cool to overnight of the iso-cholesterol (41.5 gms.) was filtered oil and the filtrate distilled to dryness. The residue was dissolved in 500 cc. of glacial acetic acid and the cholesterol isolated as in Example 6.

Yield.93 gms. M. P. 146.5-147.5 C.

As indicated above the invention is not specificall limited to the temperature ranges specified for the crystallisation from acetic or propionic acid or the selective dissolution in the same reagent, although at higher temperatures the yield is somewhat lower. The following example illustrates the yields obtained.

Example 8 Filtration Temp. Yield, Gms. Percentage 57. 7 17. 3 41. 9 l2. 6 38. 0 ll. 4 26. 9 8. 1

What I claim is:

1. Process for separating cholesterol from woolwax alcohols, characterized in that iso-cholesterol is first separated by crystallization from a solution of the raw product in a mixture of a lower hydrocarbon of the benzene series and methanol, and thereafter cholesterol is separated from waxlike impurities by selective crystallization from a. member selected from the group consisting of acetic acid and propionic acid under carefully regulated temperature conditions.

3. Process for separating cholesterol from wool Wax alcohols, characterized in that iso-cholesterol is first separated by crystallization from a solution of the raw product in a mixture of a lower hydrocarbon of the benzene series and methanol, and thereafter cholesterol is separated from wax-like impurities by selective dissolution of the impurities by a member selected from the group consisting of acetic acid and propionic acid under carefully regulated temperature conditions.

3. Process for separating cholesterol from woolwax alcohols, characterized by dissolving the alcohols in a mixture of methanol and a member selected from the group consisting of benzene, toluene, and xylene, crystallizing iso-cholesterol from said mixture, evaporating the solvent, dissolving the solid residue in a member selected from the group consisting of acetic acid and propionic acid, and selectively crystallizing cholesterol therefrom at a temperature lying within the range of 30 to 35 C.

4. Process for separating cholesterol from woolwax alcohols, characterized by dissolving the alcohols in a mixture of methanol and a member selected from the group consisting of benzene, toluene, and xylene, crystallizing iso-cholesterol from said mixture, evaporating the solvent, and selectively dissolving wax-like impurities by treatment with a member selected from the group consisting of acetic acid and propionic acid at a temperature of 30 to 35 C. to effect purification of the cholesterol.

5. Process for separating cholesterol from woolwax alcohols, characterized by dissolving the raw product in a mixture or 20 to of a member selected from the group consisting of benzene, toluene, and xylene-and to 40% methanol, cooling the solution to about 20 to 30 C. to effect crystallization of iso-cholesterol, separating the latter, evaporating the solvent from the filtrate, and separating cholesterol from wax-like impurities in the solid residue by selective treatment with a simple fatty acid at a temperature between 30 and 35 C.

6. Process for separating cholesterol from woolwax alcohols, comprising first separating isocholesterol by crystallization from a solution of the raw product in a mixture of a lower hydrocarbon of the benzene series and methanol, such crystallization being facilitated by seeding and by gradual cooling to produce relatively large crystals, and selectively separating the cholesterol from wax-like impurities by selected treatment in a solution of a simple fatty acid under carefully regulated temperature conditions.

References Cited in the file of this patent UNITED STATES PATENTS Number Name Date 2,273,046 Julian Feb. 17, 1942 2,302,67 9 Drekter Nov. 24, 1942 

1. PROCESS FOR SEPARATING CHOLESTEROL FROM WOOLWAX ALCOHOLS, CHARACTERIZED IN THAT ISO-CHOLESTEROL IS FIRST SEPARATED BY CRYSTALLIZATION FROM A SOLUTION OF THE RAW PRODUCT IN A MIXTURE OF A LOWER HYDROCARBON OF THE BENZENE SERIES AND METHANOL, AND THEREAFTER CHOLESTEROL IS SEPARATED FROM WAXLIKE IMPURITIES BY SELECTIVE CRYSTALLIZATION FROM A MEMBER SELECTED FROM THE GROUP CONSISTING OF ACETIC ACID AND PROPIONIC ACID UNDER CAREFULLY REGULATED TEMPERATURE CONDITIONS. 